Effect of Egg Size on Heat Production and the Transition of Energy from Egg to Hatchling

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1 Effect of Egg Size on Heat Production and the Transition of Energy from Egg to Hatchling A. Lourens,* 1 R. Molenaar, H. van den Brand, M. J. W. Heetkamp, R. Meijerhof, and B. Kemp *Applied Research of the Animal Sciences Group of Wageningen University and Research Centre, 8200 AB Lelystad, The Netherlands; Wageningen Institute of Animal Sciences, Adaptation Physiology Group, Wageningen University, 6700 AH Wageningen, The Netherlands; HYBRO BV, Veerstraat 38, 5830 AA, Boxmeer, The Netherlands ABSTRACT An experiment was conducted to study the needed to be set lower from d 15 onward, coinciding with effect of egg size on embryo development, heat production, and energy partitioning between egg and hatchling. Small (56.1 ± 0.12 g SEM) and large (70.0 ± 0.11 g SEM) hatching eggs were incubated in climate respiration chambers, and eggshell temperature was maintained constant at 37.8 C in both egg weight classes by adjusting machine temperature. Dry matter, ash, protein, and fat contents were determined in albumen, yolk, yolk-free body (YFB), and residual yolk (RY), and carbohydrate contents and caloric values were calculated. To achieve a constant eggshell temperature, machine temperature increased heat production in large eggs compared with small eggs. Selective nutrient uptake resulted in higher fat content and lower protein content in RY in chicks that hatched from small eggs compared with large eggs. The respiration quotient in small and large eggs was the same, and embryos in small and large eggs were equally efficient in the transfer of energy from egg to YFB. The surplus availability of nutrients in large eggs was therefore held responsible for the absolute and relative higher weight of RY in chicks that hatched from large eggs compared with small eggs. Key words: egg size, eggshell temperature, heat production, yolk-free body, residual yolk 2006 Poultry Science 85: INTRODUCTION It has been reported that during incubation, large eggs produce more heat than small eggs (Rahn et al., 1974; Vleck et al., 1980; Hoyt, 1987; Vleck and Vleck, 1987; Meijerhof and van Beek, 1993). Large eggs also face more difficulties to remove the surplus heat from the egg (French, 1997), as a result of the decreasing ratio between egg surface and egg content with increasing egg size (Vogel, 1981) and the reduced air velocity over the eggs in commercial incubators (French, 1997). If large eggs and small eggs are incubated under similar conditions, the higher heat production (HP) and increased difficulties to remove heat in large eggs will result in higher embryo temperatures in these eggs (Meijerhof and van Beek, 1993, Meijerhof, 2002). The influence of embryo temperature on embryo development and hatchability is shown by Lourens et al. (2005), who used eggshell temperature (EST) as a reflection of embryo temperature. Because egg size influences embryo temperature through heat production and heat transfer, experiments studying the effect of egg sizes on embryo 2006 Poultry Science Association, Inc. Received July 18, Accepted November 17, Corresponding author: sander.lourens@wur.nl development between species (Ricklefs, 1987) or late embryonic mortality within 1 species (Hagger et al., 1986; Reinhart and Moran, 1979) will be influenced by differences in embryo temperature, if incubator conditions are not adjusted to obtain an equal embryo temperature. To our knowledge, the effect of egg size on embryo development and hatchability is never studied independent of embryo temperature. Therefore, an experiment was conducted to incubate eggs from 2 different size classes at an equal EST. The goal of the experiment was to investigate the effect of egg size on HP, embryo development, and energy transition between egg and hatchling, when eggs of different sizes were kept on the same EST of 37.8 C throughout incubation. MATERIALS AND METHODS Experimental Setup In 4 trials, small and large eggs were incubated separately at a constant EST of 37.8 C in 1 of 2 identical climate respiration chambers. The EST from 5 individual fertile eggs was measured continuously, and machine temperature (MT) was adjusted automatically every 5 min if the median EST drifted away from 37.8 C. Heat production (HP) was calculated from oxygen consumption and carbon dioxide production. The amount of energy available in the egg albumen and egg yolk before incubation and 770

2 EGG SIZE, HEAT PRODUCTION, AND ENERGY ALLOCATION 771 the distribution of energy between the yolk-free body (YFB) and residual yolk (RY) at hatch were determined by chemical analyses. Hatching Eggs and Incubation First grade hatching eggs from 1 Hybro G grandparent stock were divided into 2 different weight classes: small ( g) and large ( g), and eggs were stored between 5 and 7 d. Per trial, 30 small eggs and 30 large eggs were incubated in 1 of 2 identical small open circuit climate respiration chambers. Additionally, from both groups, 5 eggs per trial were used to determine egg constituents. Both climate respiration chambers (267 L) contained an automatic tray turning system that turned the eggs every 30 min at an angle of 90. Two different fans mixed fresh air with recirculated air and provided a consistent airflow across the eggs. One temperature sensor (Pt100, Sensor Data BV, Rijswijk, The Netherlands) measured MT; a Vaisala sensor measured relative humidity. Another 5 sensors (Pt1000, Sensor Data BV) measured individual EST of 5 different eggs. Sensors were attached with tape (Tesa BV, Almere, The Netherlands) in heat conducting paste (Schaffner Holding AG, Switzerland) on the eggshell at the equator of the egg. All temperature sensors were compared after the experiments at different temperature levels between 36 and 40 C. Differences between individual sensors and the mean were maximal 0.1 C. Relative humidity was set at 55% constantly during the first 18 d. The EST was measured every minute, and MT was automatically adjusted according the median EST of 5 different eggs to maintain EST at 37.8 C. Eggs were candled at 7 d and 18 of incubation, and infertile eggs and eggs containing dead embryos were removed. At 18 d of incubation, all eggs were reweighed to determine egg weight loss during incubation, and eggs were transferred to hatching baskets. The EST was measured again, and MT was set at the constant value that corresponded to a constant EST of 37.8 C. For the remaining time until hatching, EST was allowed to increase. Heat Production Oxygen and carbon dioxide concentrations were measured every 9 min in both chambers and in fresh air. Carbon dioxide concentration was measured with a nondispersive infrared CO 2 analyser (type Uras 3G, Hartmann and Braun, Frankfurt, Germany). Oxygen concentration was measured with a paramagnetic oxygen analyser (type ADC7000, Analytical Development Co. Ltd., Hertfordshire, UK). The refreshed air volume was 2 L/ min during the first 18 d of incubation and 3 L/min from d 18 onward. The exact air volumes were measured with Schlumberger G1.6 dry gas meter. The HP was calculated using the formula of Romijn and Lokhorst (1961) and adjusted for fertility and embryo mortality, based on the breakout analysis described later. Table 1. Classification of true fertility and embryo mortality Time, d Characteristic 0 Unfertilized; no sign of development 1 Area vasculosa of 1 cm 2 Area vasculosa of cm 3 Blood ring/presence of subamniotic fluid 4 10 Eye present Feathers present Yolk remains outside the embryo 21 Residual yolk retracted inside the embryo; embryo ready to hatch Embryo Mortality, Hatchability, and Hatchling Measurements Clear eggs (candled at 7 d of incubation) and dead-inshell eggs (hatch debris) were opened to determine true fertility and pattern of embryonic mortality by visual appraisal. For the correction for mortality in the HP calculations, day of mortality was classified by the characteristics described in Table 1, and in more detail to the estimated day of mortality by the size of the dead embryo. The HP was expressed per living, fertile embryo (egg) based on fertility and mortality pattern. Hatchability was expressed as the percentage of chicks that hatched from the true fertile eggs, based on the breakout analysis. At 21.5 d of incubation, all hatched chicks were killed with a mixture of CO 2 and O 2. All chicks were weighed, and total chick lengths were measured by stretching chicks along a ruler and taking the length between the top of the beak and the tip of the middle toe of the right feet according to Hill (2001) or Lourens et al. (2005). Next, RY was removed and weighed. Energy Partitioning During the Transfer from Egg to Hatchling A total of 40 fresh eggs (5 eggs per trial per egg weight class) were boiled, and albumen and yolk were separated and weighed. The eggshell was dried for 24 h at room temperature and weighed. From 40 chicks per egg weight class (5 chicks per trial per egg weight class), YFB and RY were separated for analysis. Albumen, yolk, residual yolks, and the yolk-free bodies were frozen at 18 C. At a later stage, dry matter (ISO 6496, 1983), ash (ISO 5984, 1978), protein (ISO 5983, 1998), and fat contents (ISO 6492, 1999) were determined. Carbohydrate content was estimated as 1,000 ash protein fat content (grams per kilogram of DM). Finally, the energy content in the different parts was calculated using energy densities for protein, fat, and carbohydrates of respectively 16.8, 37.8, and 16.8 MJ/kg of DM (International System of Units, 1998). The efficiency in energy transfer from available energy to YFB (E YFB ) was calculated as E YFB = YFB(kJ) Albumen(kJ) + Yolk(kJ) RY (kj) 100%.

3 772 LOURENS ET AL. Statistical Analyses The nonlinear, sigmoid curves of HP and MT were analyzed in Genstat 6.1 (2002) with a restricted maximal likelihood procedure according to the following model for repeated measurements: Y ijk = +E i +T j +(ExT) ij +D k + interactions + ε ijk, where Y ijk is HP or RQ, is the overall mean, E i is egg size (i = small, large), T j is trial (j = 1,2,3,4), D k are the 21 d of incubation (k = 1,...,21), and ε ijk is the residual error term. The interaction between treatment and trial (E*T) ij represents the random effect within trial between day numbers and is used as error term to test the effects of E i and T j. A Generalized Linear Mixed model procedure for a binomial distribution with a logit link function (Genstat 6.1, 2002) was used for hatchability data. Egg- and chickrelated factors were analyzed by 2-way ANOVA with the GLM procedure of Genstat software (Genstat 6.1, 2002). In both procedures, a group of 30 eggs in 1 climate respiration chamber was used as experimental unit. The model was Y ij = +E i +T j +(E i T j )+ε ij, in which Y ij is the dependent variable, is the overall mean, E i is egg size (i = small, large), T j is trial (j = 1,2,3,4), and ε ij is the residual error term. In all analyses, nonsignificant interactions were deleted from the model. Data of DM, ash, protein, carbohydrates, and energy content were analyzed according to the latter model. Unfortunately, data of YFB and RY in trial 4 were lost, so results and conclusions with regard to energy contents and changes were based on only the first 3 trials. RESULTS Incubation Temperature and Heat Production For MT, an effect of time (P < 0.001) and an interaction between time and egg size (P = 0.032) were observed. From d 15 onward, MT needed to be decreased more compared with small eggs (Figure 1). During the course of incubation, an effect of time (P < 0.001), egg size (P = 0.001), and an interaction between time and egg size (P = 0.040) on HP were observed. The HP in large eggs was higher compared with small eggs from d 15 onward (Figure 2). Hatching Eggs, Incubation Parameters, and Hatchling Characteristics Hatching egg characteristics, incubation-related parameters, and hatchling characteristics per egg size category are summarized in Table 2. Shell weight of small eggs was lower than shell weight of large eggs, and absolute weight loss during incubation was lower in small eggs compared with large eggs. At d 18, small eggs produced 137 mw per egg (2.5 mw/g), and large eggs produced 155 mw per egg (2.2 mw/g); see Table 2. The RQ decreased between d 5 and 9 from 1.08 to 0.68, remained constant thereafter, and did not differ between small and large eggs. Embryo mortality (EM) in week 1, 2, or 3 and hatchability were similar for both egg size classes (Table 2). Chicks that hatched from small eggs weighed less and were shorter compared with chicks that hatched from large eggs. Chemical Analyses and Energy Content in Eggs and Hatchlings Egg and hatchling constituents in both egg weight classes are shown in Table 3. Fresh albumen and yolk weights were lower in small eggs compared with large eggs. Also, YFB and RY weight were lower in chicks that hatched from small eggs compared with large eggs. Dry matter content in albumen, yolk, YFB, and RY did not differ between egg size classes. Relative ash content (grams per kilogram of DM) in RY of chicks that hatched from small eggs was higher than in RY of chicks that hatched from large eggs. The RY of chicks that hatched from small eggs also contained relatively more fat (grams per kilogram of DM) than RY of chicks that hatched from large eggs. On the contrary, RY of chicks that hatched from small eggs contained relatively less protein (grams per kilogram of DM) than RY of chicks that hatched from large eggs. No differences were observed in relative carbohydrate contents (grams per kilogram of DM) in albumen, yolk, YFB, or RY between egg size classes. Also the relative energy content (megajoules per kilogram of DM) in albumen, yolk, YFB, or RY did not differ between egg size classes (Table 3). Caloric values (kilojoules in fresh product) in albumen, yolk, YFB, and RY in small and large eggs are summarized in Table 4. The total energy content in both albumen and yolk of small eggs was less than in large eggs. The total energy content of the YFB in chicks that hatched from small eggs was less than in chicks that hatched from large eggs. The total energy content of RY in chicks that hatched from small eggs was about one-half of the energy content of RY in chicks that hatched from large eggs. In albumen, the energy content of protein and carbohydrates was higher in large eggs compared with small eggs. No differences were observed in energy contents of fats. In yolk, the energy content of protein and fat was higher in large eggs compared with small eggs, and no differences were observed in energy contents of carbohydrates. In YFB and RY, the energy content of protein and fat was higher in large eggs compared with small eggs, and no differences were observed in energy contents of carbohydrates. Also the amount of energy lost during incubation from proteins and fats was higher in large eggs compared with small eggs, and no differences were observed in amount of energy lost from carbohydrates. The relative energy distribution between albumen and yolk in small and large eggs was the same: on average 21.4% of the energy was allocated in the albumen and 78.6% in the yolk. In both egg weight classes, on average 44.3% of the available energy in the egg was lost during incubation. The energy content of YFB and RY, however, differed between egg weight classes. Hatchlings that hatched from small eggs

4 EGG SIZE, HEAT PRODUCTION, AND ENERGY ALLOCATION 773 Figure 1. Machine temperature required to incubate eggs at 37.8 C eggshell temperature (EST) in small and large eggs (overall SEM = 0.06 C). contained 47.5% of the available energy in YFB compared with 43.4% in large eggs (SEM = 0.6%). The RY in hatchlings that hatched from small eggs contained 8.1% of the available energy, compared with 12.3% in hatchlings that hatched from large eggs (SEM = 1.2%). The efficiency (E yfb ) of the process of transferring energy from egg to YFB was similar between small and large eggs with regard to protein, fat, carbohydrates, and total energy. DISCUSSION Heat Production and Eggshell Temperature From the moment that large eggs produced more heat, MT needed to be decreased more for large eggs than for small eggs. In our study we observed a higher HP and lower MT in large eggs from d 15 onward, which suggests that embryonic growth in large eggs increased more from Figure 2. Heat production per egg (mw) in small and large eggs (SEM = 3.8 mw).

5 774 LOURENS ET AL. Table 2. Characteristics of hatching eggs, incubation process, and hatchlings in small and large eggs Small egg Large egg SEM Hatching egg Egg (g) 55.7 b 70.4 a 0.6 Shell (g) 6.5 b 7.8 a 0.4 Incubation Weight loss (g) 8.0 b 9.4 a 0.4 HP d 18 (mw/egg) 137 b 155 a 3.8 Fertility 1 (%) EM 2 (%) EM wk 1 (%) EM wk 2 (%) EM wk 3 (%) Hatchability 2 (%) Hatchling Chick weight (g) 36.0 b 46.4 a 0.6 Chick length (cm) 19.7 b 20.3 a 0.1 a,b Significant differences are indicated by different letters (n = 4; P < 0.05). 1 Fertility expressed as percentage of eggs set. 2 Embryo mortality (EM) and hatchability expressed as percentage of true fertile eggs. that moment onward. This is in accordance to the review by Wilson (1991), who concluded that embryo weight is not correlated with egg weight during the first half of the incubation period. If MT was not adjusted in the present study, EST would have increased more in large eggs than in small eggs from d 15 onward, as shown in Romijn and Lokhorst (1956) or calculated by Meijerhof and van Beek (1993). Adjusting MT avoids adverse effects of high EST during the last week of incubation on embryo development (Lourens et al., 2005). The HP initially increases with higher temperatures (Nichelmann et al., 1998; Janke et al., 2002), but too high temperatures decrease HP during the final stages of incubation (Janke et al., 2002). The effects of high EST on embryo development and HP can also be present in the studies of, for example, Romijn and Lokhorst (1956, 1960), Tullett and Deeming (1982), Tullett and Burton (1982), Janke et al. (2004), and O Dea et al. (2004), who incubated eggs at a constant MT of 37.8 C. In these studies, HP at 18 d of incubation ranged between 2.0 and 2.6 mw/g, comparable with the results of the present study (2.5 mw/g in small eggs and 2.2 mw/g in large eggs). Higher HP of 2.8 to 3.2 mw/g was observed by Hulet and Meijerhof (2001), who adjusted MT based on the response of the embryos on varying MT. These high values were not confirmed in the present study, which may be due to the fact that Hulet and Meijerhof (2001) used eggs of high yielding broilers, whereas in the present study eggs of grandparents of high yielding broilers were used. Breed may influence HP, as in Tona et al. (2004), in which very large differences in HP were observed between standard heavy (4.6 mw/g) and Labeltype lines (2.7 mw/g) during incubation between 17.8 and 19.5 d for 60-g eggs. However, in the study of Tona et al. (2004), eggs were incubated at a constant MT of 37.8 C, resulting in higher EST during the final stages of incubation, accelerating the timing of internal pipping in eggs from standard heavy breeders at an earlier age than in Label-type lines. This probably had a larger effect on HP than line per se. During the early stages of incubation, excess CO 2 is being washed out from the albumen (Romanoff, 1967). From d 9 onward, the RQ value in the present trial of 0.68 indicates the oxidation of fat with additional synthesis of glycogen (Beattie, 1964; Rahn, 1981). This RQ value is lower than reported by Decuypere (1984), who measured a RQ value of Glycogen is stored in liver and muscles and is mobilized during the hatching process (Bell and Freeman, 1971). Wineland et al. (2000a,b) and Wineland and Christensen (2001) concluded that incubating eggs at higher MT (38.6 vs C) early or late in incubation resulted in less utilization of the yolk and increased utili- Table 3. Weight, DM, ash, fat, protein, carbohydrate, and energy contents in albumen and yolk of small and large eggs and in yolk-free body and residual yolk of hatchlings that hatched from small and large eggs Weight 1 DM 2 Ash 3 Fat 3 Protein 3 Carbohydrates 3 Energy 4 Albumen Small eggs 32.2 b a Large eggs 41.6 a b SEM Yolk Small eggs 17.3 b Large eggs 20.8 a SEM Yolk free body Small eggs 34.0 b Large eggs 41.5 a SEM Residual yolk Small eggs 2.3 b a a b Large eggs 4.5 a b b a SEM a,b Significant differences are indicated by different letters (n = 3; P < 0.05). 1 Fresh weight (g). 2 Grams per kilogram of fresh weight. 3 Grams per kilogram of DM. 4 Megajoules per kilogram of DM.

6 EGG SIZE, HEAT PRODUCTION, AND ENERGY ALLOCATION 775 Table 4. Caloric values (kilojoules) in small and large hatching eggs (albumen and yolk), hatchlings (yolk-free body or YFB and residual yolk or RY), the amount of energy lost, and the efficiency in energy transfer between egg and YFB (E yfb ) Hatching egg (kj) Hatchling (kj) Lost E yfb Albumen Yolk YFB RY (kj) (%) Protein energy Small eggs 64.1 b 47.4 b 88.2 b 9.2 b 14.2 b 86.2 Large eggs 83.6 a 58.4 a 99.9 a 22.1 a 20.1 a 83.3 SEM Fat energy Small eggs b 65.3 b 16.4 b b 34.0 Large eggs a 76.5 a 26.0 a a 32.5 SEM Carbohydrate energy Small eggs 4.2 b Large eggs 5.4 a SEM Total energy Small eggs 68.8 b b b 26.5 b a 51.7 Large eggs 89.5 a a a 50.6 a b 49.6 SEM a,b Significant differences are indicated by different letters (n = 3; P < 0.05). zation of glycogen as energy source. High temperatures therefore not only reduce the required energy available for the embryo to emerge from the eggshell but also increase RQ. This may explain the differences in RQ observed between this experiment and the results of Decuypere (1984), who incubated at a constant MT and therefore increased EST at the end of the incubation process. When the embryo draws relatively more energy from the anaerobic system under high temperatures, HP and thus embryo development will be decreased. Energy Contents and Changes Total caloric values of eggs in the present study are comparable with data from Ar et al. (1987), who measured a caloric value of 368 kj for an average-sized hatching egg. They reported that YFB contained on average 137 kj and the RY 132 kj, so on average 99 kj was lost, which is lower than the findings in the present study. Compared with the results of Ar et al. (1987), embryos in the present study retained less energy in RY and more in YFB, produced more heat, and were therefore less efficient in energy transfer between egg and YFB. Wiley (1950) reported that embryos in small eggs find physical limitations beyond a certain size, which reduces growth and hence HP. In the present study, E yfb was similar in small eggs and large eggs, which suggests that embryos in small eggs used the available energy equally efficiently as embryos in large eggs. Still, chicks that hatched from large eggs had a larger amount of energy left in the RY. Based on similar efficiency in energy transfer, we expect that embryos in large eggs had an surplus energy reserve that was retained unused in the RY. To incubate small and large eggs at an equal EST of 37.8 C, MT profiles had to be adjusted for both size classes individually to compensate for differences in HP from d 15 onward. Embryos in small and large eggs incubated at 37.8 C were equally efficient in energy transfer from egg to YFB, and the surplus of nutrients in large eggs is likely to be responsible for the larger energy content of RY in chicks that hatched from large eggs. Relative energy density was the same in small and large eggs, but selective uptake of nutrients seemed to result in a higher fat content and lower protein content of RY in chicks that hatched from small eggs compared with large eggs. Based on these results, it can be speculated that when small and large eggs are not incubated at the same EST, HP, energy utilisation, embryo development and hatchability might have been influenced more by temperature than by egg size per se. Relations between EST and nutrient uptake, growth, and HP are largely unknown and need further investigation. ACKNOWLEDGMENTS Hybro BV (Boxmeer, The Netherlands) kindly donated the hatching eggs. The Dutch Egg and Poultry Board contributed financially to this research. The assistance of the staff in the research facilities at Zodiac, De Haar, Wageningen, The Netherlands, was greatly appreciated. REFERENCES Ar, A., B. Arieli, A. Belinsky, and Y. Yom-Tov Energy in avian eggs and hatchlings: Utilization and transfer. J. Exp. Zool. Suppl. 1: Beattie, J The glycogen content of skeletal muscle, liver and heart in late chicken embryos. Br. Poult. Sci. 5: Bell, D. J., and B. M. Freeman Physiology and biochemistry of the domestic fowl. Vol. 1. Academic Press, London, UK. Decuypere, E Incubation temperature in relation to postnatal performance in chickens. Arch. Exp. Vet. 38: French, N. A Modelling incubation temperature: The effect of incubator design, embryonic development, and egg size. Poult. Sci. 76: Genstat Genstat Release 6.1 Reference Manual. VSN Int., Wilkinson House, Oxford, UK. Hagger, C., D. Steiger-Stafl, and C. Marguerat Embryonic mortality in chicken eggs as influenced by egg weight and inbreeding. Poult. Sci. 65:

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